Glycylation regulates axonemal dyneins Physiological functions of the microtubule cytoskeleton are expected to be regulated by a variety of posttranslational tubulin modifications. For instance, tubulin glycylation is almost exclusively found in cilia and flagella, but its role in the function of these organelles remains unclear. Gadadhar et al. now demonstrate in mice that glycylation, although nonessential for the formation of cilia and flagella, coordinates the beat waveform of sperm flagella. This activity is a prerequisite for progressive sperm swimming and thus for male fertility. At the ultrastructural level, lack of glycylation perturbed the distribution of axonemal dynein conformations, which may explain the observed defects in flagellar beat. Science, this issue p. eabd4914 Loss of tubulin glycylation affects male fertility, owing to sperm motility defects, and perturbs axonemal dynein conformations. INTRODUCTION Microtubules are key components of the eukaryotic cytoskeleton. Although they are involved in a wide variety of functions, microtubules are structurally highly similar across most cell types and organisms. It was suggested that a “tubulin code,” formed by combinations of tubulin posttranslational modifications, adapts individual microtubules to specific functions within living cells. However, clear-cut functional and mechanistic data verifying this concept are still scarce. Glycylation is among the least explored posttranslational modifications of tubulin and has, so far, exclusively been found on microtubules of cilia and flagella from a variety of species. Previous work has suggested that glycylation might be essential for cilia and flagella, but mechanistic insight remains lacking. RATIONALE Two enzymes from the tubulin-tyrosine ligase-like (TTLL) family, TTLL3 and TTLL8, are essential to initiate glycylation of tubulin in mammals. To entirely abolish glycylation at the organism level and to determine its physiological function, we generated a double-knockout mouse lacking both glycylating enzymes (Ttll3−/−Ttll8−/−). Inactivation of these two enzymes led to a lack of glycylation in all analyzed cilia and flagella. This allowed us to investigate the role of glycylation in the function of these organelles. RESULTS Despite the absence of glycylation in Ttll3−/−Ttll8−/− mice, no gross defects were observed at the organism and tissue levels. Motile ependymal cilia in brain ventricles as well as motile cilia in the respiratory tract were present and appeared normal. Sperm flagella were also assembled normally, and sperm were able to swim. However, in vitro fertility assays showed that male Ttll3−/−Ttll8−/− mice were subfertile. Computer-assisted sperm analyses revealed motility defects of Ttll3−/−Ttll8−/− sperm. Further analyses showed that lack of glycylation leads to perturbed flagellar beat patterns, causing Ttll3−/−Ttll8−/− sperm to swim predominantly along circular paths. This is highly unusual for mammalian sperm and interferes with their ability to reach the oocyte for fertilization. To determine the molecular mechanisms underlying this aberrant flagellar beat, we used cryo–electron tomography. The three-dimensional structure of the 96-nm repeat of the Ttll3−/−Ttll8−/− sperm axoneme showed no aberrations in its overall assembly. By contrast, the structure of both outer and inner dynein arms (ODAs and IDAs) was perturbed in Ttll3−/−Ttll8−/− flagella. Classification analysis showed that the incidence and distribution of pre-powerstroke and post-powerstroke conformations of ODAs and IDAs were altered in Ttll3−/−Ttll8−/− sperm. These ultrastructural findings indicate that glycylation is required to efficiently control the dynein powerstroke cycle, which is essential for the generation of a physiological flagellar beat. CONCLUSION Our work shows that tubulin glycylation regulates the beat of mammalian flagella by modulating axonemal dynein motor activity. Lack of glycylation leads to perturbed sperm motility and male subfertility in mice. Considering that human sperm are more susceptible than mouse sperm to deficiencies in sperm motility, our findings imply that a perturbation of tubulin glycylation could underlie some forms of male infertility in humans. Tubulin glycylation controls sperm motility. (A) Microtubules in sperm flagella are rich in tubulin posttranslational modifications. Mice deficient for the glycylating enzymes TTLL3 and TTLL8 lack glycylation. (B) Mammalian sperm swim in linear paths. In the absence of glycylation, abnormal, mostly circular swimming patterns are observed, which impede progressive swimming. (C) Absence of glycylation leads to perturbed distribution of axonemal dynein conformations in Ttll3−/−Ttll8−/− flagella, which impedes normal flagellar beating. Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

中文翻译：

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微管蛋白糖基化控制轴索动力蛋白活性、鞭毛搏动和男性生育能力

糖基化调节轴丝动力蛋白 微管细胞骨架的生理功能预计受多种翻译后微管蛋白修饰的调节。例如，微管蛋白的糖基化几乎只存在于纤毛和鞭毛中，但其在这些细胞器功能中的作用仍不清楚。加达达尔等人。现在在老鼠身上证明，虽然糖基化对纤毛和鞭毛的形成并不重要，但它协调了精子鞭毛的跳动波形。这项活动是渐进式精子游泳的先决条件，因此也是男性生育能力的先决条件。在超微结构水平上，缺乏糖基化扰乱了轴索动力蛋白构象的分布，这可以解释观察到的鞭毛跳动缺陷。科学，本期第 3 页。eabd4914 微管蛋白糖基化的丧失会影响男性生育能力，由于精子运动缺陷，并扰乱轴索动力蛋白构象。引言 微管是真核细胞骨架的关键组成部分。尽管它们涉及多种功能，但微管在大多数细胞类型和生物体中的结构高度相似。有人提出，由微管蛋白翻译后修饰组合形成的“微管蛋白代码”使单个微管适应活细胞内的特定功能。然而，验证这一概念的明确功能和机械数据仍然很少。糖基化是研究最少的微管蛋白翻译后修饰之一，迄今为止，仅在来自各种物种的纤毛和鞭毛的微管中发现。以前的研究表明，纤毛和鞭毛可能需要糖基化，但仍然缺乏机械的洞察力。基本原理 来自微管蛋白-酪氨酸连接酶样 (TTLL) 家族的两种酶，TTLL3 和 TTLL8，对于启动哺乳动物中微管蛋白的糖基化至关重要。为了在生物体水平上完全消除糖基化并确定其生理功能，我们生成了一种缺乏两种糖化酶 (Ttll3-/-Ttll8-/-) 的双敲除小鼠。这两种酶的失活导致在所有分析的纤毛和鞭毛中缺乏糖基化。这使我们能够研究甘氨酰化在这些细胞器功能中的作用。结果尽管在 Ttll3-/-Ttll8-/- 小鼠中不存在糖基化，但在有机体和组织水平上没有观察到明显的缺陷。脑室中的活动性室管膜纤毛以及呼吸道中的活动性纤毛存在并且看起来正常。精子鞭毛也正常组装，精子能够游泳。然而，体外生育力测定显示雄性 Ttll3-/-Ttll8-/- 小鼠不育。计算机辅助精子分析揭示了 Ttll3-/-Ttll8-/- 精子的运动缺陷。进一步的分析表明，缺乏糖基化会导致鞭毛节拍模式受到干扰，导致 Ttll3-/-Ttll8-/- 精子主要沿着圆形路径游动。这对于哺乳动物精子来说是非常不寻常的，并且会干扰它们到达卵母细胞进行受精的能力。为了确定这种异常鞭毛跳动的分子机制，我们使用了低温电子断层扫描。Ttll3-/-Ttll8-/- 精子轴丝的 96 nm 重复序列的三维结构在其整体组装中没有显示出异常。相比之下，外部和内部动力蛋白臂（ODA 和 IDA）的结构在 Ttll3-/-Ttll8-/- 鞭毛中受到干扰。分类分析表明，在 Ttll3-/-Ttll8-/- 精子中，ODA 和 IDA 的动力冲程前和动力冲程后构象的发生率和分布发生了改变。这些超微结构研究结果表明，有效控制动力蛋白动力冲程循环需要糖基化，这对于产生生理鞭毛跳动至关重要。结论 我们的工作表明，微管蛋白的糖基化通过调节轴索动力蛋白的运动活动来调节哺乳动物鞭毛的节拍。缺乏糖基化会导致小鼠精子活力受到干扰和雄性不育。考虑到人类精子比小鼠精子更容易受到精子活力不足的影响，我们的研究结果表明，微管蛋白糖基化的扰动可能是人类某些形式的男性不育症的基础。微管蛋白糖基化控制精子活力。(A) 精子鞭毛中的微管富含微管蛋白翻译后修饰。缺乏糖基化酶TTLL3 和TTLL8 的小鼠缺乏糖基化。(B) 哺乳动物精子在线性路径中游动。在没有糖基化的情况下，观察到异常的、主要是圆形的游泳模式，这阻碍了渐进式游泳。(C) 不存在糖基化导致 Ttll3-/-Ttll8-/- 鞭毛中轴丝动力蛋白构象的分布受到干扰，这阻碍了正常的鞭毛跳动。微管细胞骨架的翻译后修饰已成为细胞功能的关键调节因子，它们的扰动与越来越多的人类病理有关。微管蛋白糖基化修饰纤毛和鞭毛中的微管，但其功能和机制作用仍不清楚。在这项研究中，我们生成了一个完全缺乏微管蛋白糖基化的小鼠模型。雄性小鼠由于精子鞭毛的异常跳动模式而无法生育，这阻碍了精子细胞的直线游动。使用冷冻电子断层扫描，我们发现缺乏糖基化导致精子轴突内动力蛋白臂的构象异常，为观察到的功能障碍提供了结构基础。我们的研究结果揭示了微管糖基化对控制鞭毛跳动、定向精子游动和男性生育能力的重要性。我们生成了一个完全缺乏微管蛋白糖基化的小鼠模型。雄性小鼠由于精子鞭毛的异常跳动模式而无法生育，这阻碍了精子细胞的直线游动。使用冷冻电子断层扫描，我们发现缺乏糖基化导致精子轴突内动力蛋白臂的构象异常，为观察到的功能障碍提供了结构基础。我们的研究结果揭示了微管糖基化对控制鞭毛跳动、定向精子游动和男性生育能力的重要性。我们生成了一个完全缺乏微管蛋白糖基化的小鼠模型。雄性小鼠由于精子鞭毛的异常跳动模式而无法生育，这阻碍了精子细胞的直线游动。使用冷冻电子断层扫描，我们发现缺乏糖基化导致精子轴突内动力蛋白臂的构象异常，为观察到的功能障碍提供了结构基础。我们的研究结果揭示了微管糖基化对控制鞭毛跳动、定向精子游动和男性生育能力的重要性。我们发现，缺乏糖基化导致精子轴突内动力蛋白臂的构象异常，为观察到的功能障碍提供了结构基础。我们的研究结果揭示了微管糖基化对控制鞭毛跳动、定向精子游动和男性生育能力的重要性。我们发现，缺乏糖基化导致精子轴突内动力蛋白臂的构象异常，为观察到的功能障碍提供了结构基础。我们的研究结果揭示了微管糖基化对控制鞭毛跳动、定向精子游动和男性生育能力的重要性。